This project will explore the mechanisms that govern chromosome segregation in the final stages of cell division. This process is triggered by a poorly understood enzyme called the Anaphase-Promoting Complex or APC. The APC is a large, multisubunit ubiquitin ligase that catalyzes the ubiquitination of several key regulators of late mitotic events. In the proposed studies, biochemical approaches will be used in the budding yeast Saccharomyces cerevisiae to dissect the enzymological mechanisms by which the APC recognizes its substrates and assembles polyubiquitin chains that direct those substrates to the proteasome for destruction. Three specific aims are proposed. The first aim focuses on the important question of how the APC recognizes its substrates: peptide crosslinking methods will be used to identify binding sites on the APC for substrate and activator subunits, and the mechanisms by which activator subunits promote catalysis will be explored, in part through the reconstitution and analysis of a 3-subunit core APC module. The second aim centers on the regulation of APC activity by components of the spindle assembly checkpoint. Preliminary data indicate that these components promote autoubiquitination of the APC activator subunit, and in the proposed studies this self-regulatory mechanism will be explored with purified components in vitro. Finally, the third aim addresses the collaboration between the APC and its associated ubiquitin-conjugating enzymes or E2s. Previous work demonstrated that an E2 called Ubc1 works with the APC to assemble polyubiquitin chains on APC targets, and the proposed studies will address the mechanisms by which Ubc1 is recruited to the APC to drive chain assembly. The knowledge gained from these studies will provide new insights into the control of chromosome segregation - errors in which often contribute to developmental problems and cancer progression. These studies are also likely to illuminate general mechanisms of protein ubiquitination, a regulatory modification of major importance throughout cell biology and human disease. PUBLIC HEALTH RELEVANCE: When a cell reproduces, the chromosomes are first duplicated and then segregated into a pair of daughter cells. Errors in this process can result in genetic damage or defects in chromosome number, which can accelerate cancer progression or cause developmental defects. The proposed studies focus on a poorly understood enzyme called the anaphase-promoting complex, which serves as a critical component of the regulatory system that governs the final steps in cell division. These studies will lead to a better understanding of how errors in chromosome segregation can arise in human disease.